An acellular nerve scaffold fabricated by hypotonic buffer combined with freeze-drying

Abstract

Objective To explore the feasibility of fabricating an acellular nerve scaffold by the technique of hypotonic buffer combined with freeze-drying.Methods Fresh rabbit sciatic nerves deprived of surrounding connective tissue were prepared and divided into 3 groups:non-treatment group where the fresh nerve received no treatment,conventional group where the acellularization process was completed by Hudsom method,and modified group where the acellularization process was completed by the technique of hypotonic buffer combined with freeze-drying.After the acellularization process,the scaffolds were investigated by histological staining,SEM observation,interval porosity and void diameter measurement,biomechanical test,and biocompatibility analysis.MTT assay was done to access the cytotoxicity of scaffold leaching liquor.Rabbit adipose-derived stem cells (ADSCs) were isolated and seeded into the modified scaffold.Cell viability was analyzed using HE staining and dead/live assay.Results Compared with the non-treatment and conventional groups,in the modified group the blue-stained nucleoli disappeared and only the basement membrane was observed of Schwann cells which were loosely disposed in waves.Laminin immunofluorescence was positive in all groups.Compared with the non-treatment group,in the acellular nerve scaffolds in the conventional and modified groups,the myelin sheaths,axons and cells all disappeared and the vessels of basilar membrane paralleled in longitudinal sections,but the structure of the modified scaffold was looser.The interval porosity (49.34％ ± 9.76％) and void diameter (17.61 μm ± 3.69 μm) of the modified scaffold were significantly larger than those (34.50％ ± 7.26％ and 11.96 μm ± 3.10 μm) of the conventional scaffold (P ＜ 0.05).The differences regarding biomechanical properties were not statistically significant between the 3 groups (P ＞ 0.05).MTT showed no significant differences in the effects of leaching liquor and control culture medium of different concentrations on the cellular proliferation (P ＞ 0.05).HE staining and dead/live assay showed fine cellular viability in the modified scaffold.Conclusion The improved acellular nerve scaffold fabricated by the technique of hypotonic buffer combined with freeze-drying may be a proper cell-carrier candidate for nerve tissue engineering,because it is characterized by good pore diameter and porosity,appropriate biomechanical character,non-toxicity and good biocompatibility. Key words: Tissue engineering; Tissue scaffolds; Biomechanics; Freeze-drying; Hypotonic buffer